Phosphoric acid, mono- and di-isotridecyl esters, compd. with 2,2'-iminobis[ethanol]

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From May 15, 2017 to 31 May, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

yes

Remarks:

see 'Principles of method if other than guideline'

Principles of method if other than guideline:

- Analyses conducted to support the information cited in the Certificate of Analysis or equivalent document for the test substance were not conducted in compliance with the GLP or Good Manufacturing Practice (GMP) regulations. The characterization of the test substance was conducted under a Sponsor or Sponsor subcontractor quality system.

- Concentration, stability, and homogeneity of test substance formulations were not determined in this study. However, to limit the impact, the test substance preparation was performed with approved procedures and documented in detail. Preparations were visually inspected for homogeneity prior to use and all preparations were used within 4 hours after preparation of the formulation.

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

KeratinoSensTM assay is validated in vitro skin sensitization tests and is recommended in international guidelines (e.g. OECD).

Specific details on test material used for the study:

Batch: RE 10-7; Appearance: lightly yellow paste; Purity: 92.75 %; Purity/composition correction factor: based on purity = 1.078.

Details on the study design:

TEST SYSTEM

A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Switserland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

CELL CULTURE

Basic medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Maintenance medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/ml).

Exposure medium
Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 59 - 92 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Subculturing
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages.

EXPERIMENTAL DESIGN

Plating of Cells
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was 21, 16 and 24 in experiment 1, 2 and 3, respectively.

Reatment of Cells
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about
48 hours at 37±1.0 C in the presence of 5% CO2. In total 3 experiments were performed. In addition 3 experiments were rejected due to a technical error or since not all acceptability criteria were met.

Luciferase Activity Measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time one second).

Cytotoxicity Assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured with the TECAN Infinite® M200 Pro Plate Reader.

Positive control results:

The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol (EDMG, Sigma, The Netherlands), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted, so that the final concentration of the positive control ranges from 7.81 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.

Acceptance criteria for the positive criteria were met:
- The luciferase activity induction obtained with the positive control, ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 µM (17.5 µM, 37.5 µM and
30.6 µM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (28.1-fold, 3.59-fold and 2.76-fold in experiment 1, 2 and 3, respectively).

Key result

Parameter:

other: IC50 in µg/mL

Value:

8.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

EC1.5 = 17.5 µM

Key result

Parameter:

other: IC50 in µg/mL

Value:

17.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

EC1.5 = 37.5 µM

Key result

Parameter:

other: IC50 in µg/mL

Value:

11.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

EC1.5 = 30.6 µM

Other effects / acceptance of results:

Experiment 1
• The test isubstance showed toxicity. The calculated IC30 was 6.6 µg/mL and the calculated IC50 was 8.3 µg/mL.
• No luciferase activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test substance. The Imax was 0.61 and therefore no EC1.5 could be calculated.
• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 28.1 and the EC1.5 17.5 µM.

Experiment 2
• The test substance showed toxicity. The calculated IC30 was 14.9 µg/mL and the calculated IC50 was 17.8 µg/mL.
• A luciferase activity induction was observed after treatment with the test substance at 12.5 µg/mL only. The Imax was 2.07 and the EC1.5 9.4 µg/mL.
• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.59 and the EC1.5 37.5 µM.

Experiment 3
• The test substance showed toxicity. The calculated IC30 was 9.4 µg/mL and the calculated IC50 was 11.9 µg/mL.
• No luciferase activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test substance. The Imax was 1.00 and therefore no EC1.5 could be calculated.
• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.76 and the EC1.5 30.6 µM.

All three tests passed the acceptance criteria.

Data interpretation:

A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:

1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test).

2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration).

3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW).

4. There is an apparent overall dose-response for luciferase induction.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance is classified as negative in the KeratinoSens assay (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).

Executive summary:

A study was conducted to determine the ability of the substance to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay according to OECD 422D Guideline, in compliance with GLP. A correction factor of 1.078 was used to correct for the purity (92.75%) of the test substance. The test substance was dissolved in dimethyl sulfoxide at 40 mg/mL and from this stock 11 spike solutions in DMSO were prepared. Three independent experiments were performed. The cells were incubated with the test substance at concentration ranges of 0.195 - 400 µg/mL, 0.024 - 50 µg/mL and 4.29 - 50 µg/mL for 48 h in experiment 1, 2 and 3 respectively. The activation of the ARE-dependent pathway was assessed by measuring the luciferase activity induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay. The test substance showed toxicity in all three experiments and the IC50 values were 8.3 µg/mL, 17.8 µg/mL and 11.9 µg/mL in experiment 1, 2 and 3, respectively. In the first and the third experiments, no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. In the second experiment, an induction of luciferase activity (EC1.5 value 9.4 µg/mL; p<0.001 Student’s t test) was observed. The maximum luciferase activity induction (Imax) was 2.07 -fold at 12.5 µg/mL. Under the study conditions, the substance was classified as negative in the KeratinoSens assay (Westerink, 2017).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From April 20, 2017 to May 16, 2017

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals No. 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

OECD (2016)

Deviations:

not specified

GLP compliance:

yes

Type of study:

other: Changes in CD86 cell surface marker expression on a human histiocytic lymphoma cell line, U937 cells

Details on the study design:

PRINCIPLE OF THE STUDY: The h-CLAT assay measures markers of dendritic cell (DC) activation in the human leukaemia cell line THP-1. The ability of test substance to induce activation of CD86 and CD54 in THP-1 cells is used as an assessment of the chemical’s skin sensitisation potential. Activation of CD54 and CD86 protein markers was measured by flow cytometry following 24 h exposure to the test substance.

DOSE FINDING ASSAY
A solubility assessment and two independent dose finding assay (determining CV75 value i.e. the estimated concentration yielding 75% cell viability) were performed, using doses of test substance between 0.008 mg/mL and 1 mg/mL in saline.

TEST SYSTEM: The test system was THP-1 human monocytic leukaemia cells (ATCC® catalogue no. TIB 202, batch no. 62996831).

Positive control results:

The study was terminated prior to its completion

Key result

Run / experiment:

other: Mean of two run

Parameter:

other: CV75 (mg/mL)

Remarks:

A concentration showing 75% of THP-1 cell survival (25% cytotoxicity)

Value:

0.041

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

not determinable

Remarks:

The study was terminated prior to its completion, due to technical difficulties related to Test facility

Other effects / acceptance of results:

The study was terminated prior to its completion

Dose Finding Assay

• Test substance was soluble at a concentration of 104 mg/mL in saline.
• The mean CV₇₅ value calculated was 0.041 mg/mL.

Results of h-CLAT Assay:

• The study was terminated prior to its completion, due to technical difficulties with the h‑CLAT assay. These technical difficulties were not due to incompatibility of the test substance with the test system, but instead related to the Test Facility being no longer able to perform the assay, independent of the test substance.
• A second acceptable h‑CLAT assay was not conducted, therefore no prediction of the sensitizing potential of test substance was made.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the study conditions, h-CLAT assay was not completed due to technical difficulties related to test facility, therefore no prediction can be made regarding skin sensitisation potential of test substance.

Executive summary:

A study was conducted to determine the skin sensitisation potential of test substance, using the in vitro human Cell Line Activation Test (h-CLAT) method, according to OECD 442E. The study was terminated prior to its completion. One h-CLAT assay was performed but a second assay could not be performed. Under the study conditions, h‑CLAT assay was not completed due to technical difficulties related to test facility, therefore no prediction can be made regarding skin sensitisation potential of test substance (Vinall, 2017).

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From October 18, 2017 to December 22, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitisation: U937 cell line activation Test)

Version / remarks:

OECD 442E – Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test (U- SENS™)’ (9 October 2017)

Deviations:

no

GLP compliance:

yes

Type of study:

other: Drift in CD86 cell surface marker expression on a human histiocytic lymphoma cell line, U937 cells

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended for skin sensitization to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the U-SENS assay, which is recommended in international guideline (e.g. OECD).

Details on the study design:

PRINCIPLE OF TEST:
The U937 cell line activation Test (U-SENS™) method is an in vitro assay that quantifies changes of CD86 cell surface marker expression on a human histiocytic lymphoma cell line, U937 cells, following 45±3 h exposure to the test substance. The CD86 surface marker is one typical marker of U937 activation. CD86 is known to be a co-stimulatory molecule that may mimic monocytic activation, which plays a critical role in T-cell priming. The changes of CD86 cell surface marker expression are measured by flow cytometry following cell staining typically with fluorescein isothiocyanate (FITC)-labelled antibodies. Cytotoxicity measurement is also conducted [e.g. by using propidium iodide (PI)] concurrently to assess whether upregulation of CD86 cell surface marker expression occurs at sub-cytotoxic concentrations. The stimulation index (S.I.) of CD86 cell surface marker compared to solvent/vehicle control is calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.

TEST SYSTEM:
U937 human monocytes (Inducible CD86-expressing cells)
Source: ATCC (American Type Culture Collection, Virginia, USA). ATCC no.: CRL-1593.2TM.
- Stock cultures of these cells were stored in liquid nitrogen (-196°C). Cells were used after an acclimatisation period of approximately 8 days after thawing and were not sub-cultured more than 21 times. Once a year the cell line were checked for infection with a mycoplasma detection test. Each batch of cells received from a supplier are submitted to a qualification process to guarantee their suitability (spontaneous CD86 level) for the test by comparison with the historical data or data from the literature.

REFERENCE SUBSTANCES:
- Negative Control: Lactic Acid (LA, RS471; Batch# BCBK8387V) was used as negative control. On the treatment day, a solution at 10 mg/mL was prepared in culture medium. This solution was diluted 1:25 in order to obtain a 0.4 mg/mL stock solution (final dose level 200 µg/mL).
- Positive Control: 2,4,6-Trinitrobenzenesulfonic acid (TNBS; RS599; Batch# BCBR6376V) was provided as 1 M solution. On the treatment day a 10 mg/mL solution was prepared. This solution was diluted 1:100 in order to obtain a 0.1 mg/mL stock solution (final dose level 50 µg/mL).
- Vehicle: 0.40 % dimethyl sulfoxide (DMSO, Sigma, Zwijndrecht, The Netherlands) in Cell Culture medium (RPMI-1640, Life Technologies, Bleiswijk, The Netherlands).

CELL CULTURE MEDIUM:
Stock and treatment cultures were performed in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (FCS), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively).

ENVIRONMENTAL CONDITIONS
All incubations were carried out in a humid atmosphere of 80 - 100% (actual range 28 –
101 %) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.6 – 47.3°C). Based on laboratory historical data these deviations are considered not to affect the study integrity.

DOSE LEVELS:
A solubility test was performed. The test substance formed a non-homogenous suspension in complete medium at 0.50 mg/mL. The test substance was dissolved in DMSO to a final concentration of 0.50 mg/mL and formed clear solution.

In the main experiments the test substance was suspended in dimethyl sulfoxide (DMSO) at
50 mg/mL. The stock was diluted to the following final test concentrations:
- Experiment 1: 200, 100, 50, 20, 10 and 1 µg/mL
- Experiment 2: 200, 100, 50, 35, 20 and 10 µg/mL
- Precipitation was observed at the end of the incubation period in the 96-well plates in experiment 2 at test concentrations of 50, 100 and 200 µg/mL.
- No correction was made for the composition/purity of the test substance.

REPLICATES:
All assays were performed using two replicate culture-wells for the test substance. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding. Three replicates of complete medium untreated control, solvent/vehicle control, negative and positive controls were tested.

TREATMENT OF CELLS:
Cultures were initiated in 96-well plates using 100 µL/well of a cell suspension adjusted at 5.0 x 105 viable cells/mL. If the cell viability is < 90% the cells were not used. Cells are treated for 45 ± 3 h with the selected doses. A negative untreated control (culture medium), a vehicle control and the positive and negative control items were included. The final volume in the wells was 200 µL.
Precipitate evaluation: Before and after 45 ± 3 h of exposure, wells were checked for precipitate. If any, precipitate is documented in the raw data and reported in the table of the results.

CELL ANTIBODIES STAINING FOR IgG1 and CD86:
Treated Cells are washed, re-suspended with buffer and stained with below Fluorescein Isothiocyanate (FITC)-conjugated antibodies (used for both IgG1 and CD86 staining), by incubating refrigerated in the dark for 30 minutes
- Mouse IgG1 of unknown specificity, for isotypic control
- Human CD86 specific mouse IgG1

FLOW CYTOMETRY ANALYSIS:
- Expression level of CD86 and cell viability are analysed using flow cytometry.
- For each culture (IgG1 well and CD86 well), the percentage of viable cells (PI negative cells) was evaluated. The viability for each dose level is the mean of the IgG1 well and CD86 well. The theoretical concentration at which the chemical induces 30% cytotoxicity (i.e., 70% viability) denoted as 'CV70'; and The stimulation index (S.I.) of CD86 was calculated.
- Refer to the section 'Any other information on materials and methods incl. tables' for calculation details

Positive control results:

Experiment 1: The positive control (TNBS) showed a S.I. ≥461% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Experiment 2: The positive control (TNBS) showed a S.I. ≥176% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
- Both tests passed the acceptance criteria

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC150 (Test substance concentration (µg/mL) that induces CD86 stimulation index (S.I.) of 150%)

Value:

19

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Test substance cytotoxicity concentration for 70% viability (CV70) was 38 µg/mL

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: EC150 (Test substance concentration (µg/mL) that induces CD86 stimulation index (S.I.) of 150%)

Value:

21

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Test substance cytotoxicity concentration for 70% viability (CV70) was 25 µg/mL

Other effects / acceptance of results:

Experiment 1: The negative control (LA) showed a S.I. <70% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Experiment 2: The negative control (LA) showed a S.I. <97% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Both tests passed the acceptance criteria:

- At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (96% in experiment 1 and 95% in experiment 2).
- The mean viability of the triplicate DMSO vehicle control cells was above the threshold of 90% (99% in experiment 1 and 97% in experiment 2).
- The DMSO vehicle control mean value of its triplicate CD86 S.I. was smaller than 250% of the mean of the triplicate CD86 S.I. of untreated U937 cells in both experiments.
- The CD86 basal expression of untreated U937 cells is within the range of ≥2% and ≤25%.
- At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥0.6% and <1.5% in both experiments.
- No drift in CD86 expression was observed in the untreated controls and negative controls.

- Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

EXPERIMENT 1

• No precipitation was observed at the end of the incubation period in the 96-well plates.
• The test substance showed only toxicity at 50 µg/mL, however at 100 and 200 µg/mL the acquired cells were so low, due to toxicity, that no accurate viability measurement was obtained. Therefore toxicity was assumed at these concentrations as well and the calculated CV70 was 38 µg/mL.
• A biologically relevant increase in the expression of CD86 was observed after treatment with the test substance and the EC150 was 19 µg/mL.

EXPERIMENT 2

• Precipitation was observed at the end of the incubation period in the 96-well plates at 50, 100 and 200 µg/mL.
• The test substance showed toxicity and the calculated CV70 was 25 µg/mL.
• A biologically relevant increase in the expression of CD86 was observed after treatment with the test substance, and the EC150 was 21 µg/mL.

The test substance showed toxicity (CV₇₀ values of 38 µg/mL and 25 µg/mL in experiment 1 and 2, respectively). A biologically relevant induction of the CD86 activity (EC₁₅₀ values of 19 µg/mL and 21 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The test substance is classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed with EC₁₅₀ values < CV₇₀ values.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the study condition, test substance was determined to be skin sensitiser

Executive summary:

A study was conducted to determine the skin sensitisation potential of test substance, using the U937 cell line activation test method, according to OECD 442E, in compliance with GLP. The U937 cell line activation Test (U-SENS™) method is an in vitro assay that quantifies changes of CD86 cell surface marker expression on a human histiocytic lymphoma cell line, U937 cells, following 45±3 h exposure to the test substance. The cells in this experiment were incubated with the test substance in a concentration range of 1.0 – 200 µg/mL and 10 – 200 µg/mL in experiment 1 and 2, respectively. The increase of CD86 cell surface marker expression was assessed by measuring the amount of fluorescent cell staining of the CD86 cell surface marker compared to the vehicle control. In addition, the viability was assessed with propidium iodide. Results were derived based on the CV70 value (i.e. a test substance concentration showing 70% of U937 cell survival) and the EC150 value (i.e. the concentration at which the test substance induces a CD86 stimulation index (S.I.) of 150%). In both experiments the positive and negative control were considered valid. Overall it was concluded that the test conditions were adequate and that the test system functioned properly. The test substance showed toxicity (CV70 values of 38 µg/mL and 25 µg/mL in experiment 1 and 2, respectively). A biologically relevant induction of the CD86 activity (EC150 values of 19 µg/mL and 21 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The test substance was classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed with EC150 values < CV70 values. Under the study condition, test substance was determined to be a skin sensitiser (Eurlings, 2018).

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Dec. 11, 2017 to Apr. 03, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC No 440/2008, part B. “Skin Sensitization: Guinea-Pig Maximization Test (GPMT)”

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The Guinea Pig Maximization Test was selected since the test substance is a surfactant and the Local Lymph Node Assay as preferred alternative has shown to provide false positive results for surfactants

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle, France; Charles River Deutschland, Sulzfeld, Germany
- Age at the Initiation of Dosing: Young adult animals (approximately 4 weeks old).
- Weight at the Initiation of Dosing: 258 to 289 g.
- Acclimation: 5 d before the commencement of dosing.
- Housing: Up to 5 animals of the same sex and same dosing group together in labeled Noryl cages containing sterilized sawdust as bedding material
- Diet: Complete maintenance diet for guinea pigs (MS-H, SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum
- Water: Municipal tap-water was freely available to each animal via water bottles.
- Other: Nulliparous and non-pregnant animals were included in the study

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24°C
- Humidity: 40 to 70%
- Air changes: Ten or greater air changes per hour with 100% fresh air
- Photoperiod: 12 hour light/12 hour dark cycle

ANIMAL ENRICHMENT: For psychological/environmental enrichment, animals were provided with shelters, Play tunnels, Datesand, except when interrupted by study procedures/activities.

No. of animals per dose:

Experimental group: 10 females
Control group: 5 females

Details on study design:

VEHICLE: Water (Elix, Millipore S.A.S., Molsheim, France).

PREPARATION OF TEST SUBSTANCE
Test substance dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.
Any residual volumes were discarded.
Analysis of test substance in vehicle for concentration, stability, homogeneity was not performed.

ANIMAL SELECTION, IDENTIFICATION AND ASSIGNMENT TO EXPERIMENTAL GROUPS
- Animal Identification: At study assignment, each animal was identified using an ear tattoo.
- Assignment to experimental groups: Animals were assigned to the study at the discretion of the coordinating biotechnician according to body weights, with all animals within ± 20% of the sex mean. Animals in poor health or at extremes of body weight range were not assigned to the study.
- Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main study. The selection of concentrations was based on the following criteria:
- The concentrations are well-tolerated systemically by the animals.
- For the induction exposures: the highest possible concentration that produced mild to moderate irritation (grades 2 - 3).
- For challenge exposure: the maximum non-irritant concentration.
Series of test substance concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 100%, 50%, 20%, 10%, 5%, 2%, 1% and if needed, further lower concentrations using the same steps.
The test system and procedures were identical to those used during the main study, unless otherwise specified. The six animals selected were between 4 and 9 weeks old. No body weights were determined.

- Intradermal injections:
Initially, a series of four test substance concentrations was tested; the highest concentration was the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The resulting dermal reactions were assessed 24 and 48 h after treatment.
Based on the results in the initially treated animals, two additional animals were treated in a similar manner with four lower concentrations at a later stage.

- Epidermal application:
A series of four test substance concentrations was tested; the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each or an equivalent amount when dosed with a spatula) per animal to the clipped flank, using Metalline patches# (2x3 cm) mounted on Medical tape, which were held in place with Micropore tape and subsequently Coban elastic bandage. The initially used animals receiving intradermal injections were treated with the lowest concentrations and two other animals with the highest concentrations. After 24 h, the dressing was removed and the skin cleaned of residual test substance using water.
The resulting dermal reactions were assessed for irritation 24 and 48 h after removal of the dressings.

MAIN STUDY
The concentrations and induction method were selected based on the results of the preliminary irritation study.

A. INDUCTION EXPOSURE (Experimental animals)
- Day 1: The scapular region was clipped and three pairs of intradermal injections (0.1 mL/site) were made in this area as follows:
a) A 1:1 w/w mixture of Freunds' Complete Adjuvant (Sigma-Aldrich, Steinheim, Germany) with water for injection (Fresenius AG, Bad Homburg, Germany).
b) The test substance at a 2% concentration.
c) A 1:1 w/w mixture of the test substance, at twice the concentration used in (b) and Freunds' Complete Adjuvant.
Note: One of each pair was on each side of the midline and from cranial a) to caudal c).
- Day 3: The dermal reactions caused by the intradermal injections were assessed for irritation.
- Day 7: The scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl-sulfate (SDS, Boom, Meppel, The Netherlands) in vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction.
- Day 8: The 10% SDS treated area between the injection sites was treated with 0.5 mL or an equivalent amount when dosed with a spatula of a 100% test substance concentration using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.
- CONTROL ANIMALS: The control animals were treated as described for the experimental animals except that, instead of the test substance, the vehicle was administered.

B. CHALLENGE EXPOSURE (Experimental and Control animals)
- Day 21: One flank of all animals was clipped and treated by epidermal application of a 100% test substance concentration and the vehicle (0.1 mL each, using Patch Test Plasters (Curatest F®, Lohmann, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage.
- The dressing was removed after 24 h exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 h after removal of the dressing.

TERMINATION
- After termination, animals were sacrificed using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and an intra-peritoneal injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

Challenge controls:

Control animals were treated similar to test animals in 'CHALLENGE EXPOSURE'. For details refer to the section 'Details on study design'

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100% test substance concentration

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions were noted for any animal 24 h after exposure

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100% test substance concentration

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions were noted for any animal 48 h after exposure

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100% test substance concentration

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100% test substance

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Remarks on result:

not measured/tested

PRELIMINARY IRRITATION STUDY

Based on the results, the test substance concentrations selected for the main study were a 2% concentration for the intradermal induction and a 100% concentration for the epidermal induction exposure. Only very slight erythema was observed to the highest test substance concentration epidermally tested. Therefore, the test site of all animals of the main study were treated with 10% SDS approximately 24 h before the epidermal induction, to provoke a mild inflammatory reaction.

A 100% test substance concentration was selected for the challenge phase.

MAIN STUDY

INDUCTION PHASE: The reactions noted in the experimental and control animals after the epidermal induction exposure were considered to be enhanced by the SDS treatment.

CHALLENGE PHASE: No skin reactions were evident after the challenge exposure in the experimental and control animals.

TOXICITY / MORTALITY: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

There was no evidence that test substance caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 100% test substance concentration in the challenge phase.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, test substance was determined to be non-sensitising.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance using the Guinea-Pig Maximisation Test method (GPMT) according to OECD Guideline 406, in compliance with GLP. Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, 10 experimental animals were intradermally injected with a 2% concentration and epidermally exposed to a 100% concentration (undiluted). Five control animals were similarly treated, but with vehicle alone. Approximately 24 h before the epidermal induction exposure, all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were epidermally challenged with undiluted test substance (100% concentration) and the vehicle. There was no evidence that the test substance caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental or control animals in response to a 100% test substance concentration in the challenge phase. No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study. Under the study conditions, the test substance was determined to be non-sensitising (Van Sas, 2018).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

KeratinoSens assay:

A study was conducted to determine the ability of the substance to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay according to OECD 422D Guideline, in compliance with GLP. A correction factor of 1.078 was used to correct for the purity (92.75%) of the test substance. The test substance was dissolved in dimethyl sulfoxide at 40 mg/mL and from this stock 11 spike solutions in DMSO were prepared. Three independent experiments were performed. The cells were incubated with the test substance at concentration ranges of 0.195 - 400 µg/mL, 0.024 - 50 µg/mL and 4.29 - 50 µg/mL for 48 h in experiment 1, 2 and 3 respectively. The activation of the ARE-dependent pathway was assessed by measuring the luciferase activity induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay. The test substance showed toxicity in all three experiments and the IC50 values were 8.3 µg/mL, 17.8 µg/mL and 11.9 µg/mL in experiment 1, 2 and 3, respectively. In the first and the third experiments, no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. In the second experiment, an induction of luciferase activity (EC1.5 value 9.4 µg/mL ; p<0.001 Student’s t test) was observed. The maximum luciferase activity induction (Imax) was 2.07 -fold at 12.5 µg/mL. Under the study conditions, the substance was classified as negative in the KeratinoSens assay (Westerink, 2017).

h-CLAT assay:

A study was conducted to determine the skin sensitisation potential of test substance, using thein vitrohuman Cell Line Activation Test (h-CLAT) method, according to OECD 442E. The study was terminated prior to its completion. One h-CLAT assay was performed but a second assay could not be performed. Under the study conditions, h‑CLAT assay was not completed due to technical difficulties related to test facility, therefore no prediction can be made regarding skin sensitisation potential of test substance (Vinall, 2017).

U973 assay:

A study was conducted to determine the skin sensitisation potential of test substance, using the U937 cell line activation test method, according to OECD 442E, in compliance with GLP. The U937 cell line activation Test (U-SENS™) method is anin vitroassay that quantifies changes of CD86 cell surface marker expression on a human histiocytic lymphoma cell line, U937 cells, following 45±3 h exposure to the test substance. The cells in this experiment were incubated with the test substance in a concentration range of 1.0 – 200 µg/mL and 10 – 200 µg/mL in experiment 1 and 2, respectively. The increase of CD86 cell surface marker expression was assessed by measuring the amount of fluorescent cell staining of the CD86 cell surface marker compared to the vehicle control. In addition, the viability was assessed with propidium iodide. Results were derived based on the CV70 value (i.e. a test substance concentration showing 70% of U937 cell survival) and the EC150 value (i.e. the concentration at which the test substance induces a CD86 stimulation index (S.I.) of 150%). In both experiments the positive and negative control were considered valid. Overall it was concluded that the test conditions were adequate and that the test system functioned properly. The test substance showed toxicity (CV70 values of 38 µg/mL and 25 µg/mL in experiment 1 and 2, respectively). A biologically relevant induction of the CD86 activity (EC150 values of 19 µg/mL and 21 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The test substance was classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed with EC150 values < CV70 values. Under the study condition, test substance was determined to be a skin sensitiser (Eurlings, 2018).

Guinea pig maximisation test (GPMT):

A study was conducted to determine the skin sensitisation potential of the test substance using the Guinea-Pig Maximisation Test method (GPMT) according to OECD Guideline 406, in compliance with GLP. Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, 10 experimental animals were intradermally injected with a 2% concentration and epidermally exposed to a 100% concentration (undiluted). Five control animals were similarly treated, but with vehicle alone. Approximately 24 h before the epidermal induction exposure, all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were epidermally challenged with undiluted test substance (100% concentration) and the vehicle. There was no evidence that test substance caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental or control animals in response to a 100% test substance concentration in the challenge phase. No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study. Under the study conditions, the test substance was determined to be non-sensitising (Van Sas, 2018).

Justification for classification or non-classification

Based on the results of Guinea Pig Maximisation Test, the substance does not need to be classified for sensitisation according to EU CLP (EC 1272/2008) criteria.